MMTV-SV40-Spy1A and Spy1A-pTRE Transgenic Mouse Models

ABSTRACT

In one aspect, the invention provides a transgenic non-human animal model having germ cells and somatic cells containing an endogenous MMTV-SV40-Spy1A gene sequence introduced into said animal model or an ancestor of said animal model at an embryonic stage, wherein said gene sequence comprises a mouse mammary tumor virus gene (MMTV), a functionally disrupted SV40 gene (SV40) and a human Spy1A gene. In another aspect, the present invention provides a transgenic non-human animal model whose germ cells and somatic cells contain an endogenous Spy1A-pTRE-Tight gene sequence introduced into said animal model or an ancestor of said animal model at an embryonic stage. Preferably, the Spy1A-pTRE-Tight animal model expresses the Spy1A gene and develop cancer, preferably breast cancer, when administered with tetracycline, preferably doxycycline.

RELATED APPLICATIONS

This application is a continuation application of earlier U.S.application Ser. No. 14/679,482 filed 6 Apr. 2015, which is acontinuation-in-part application of U.S. application Ser. No. 13/987,769filed 30 Aug. 2013, which claims the benefit of 35 U.S.C. 119(e) to U.S.Provisional Application Ser. No. 61/695,719 filed on 31 Aug. 2012 andU.S. Provisional Application Ser. No. 61/743,501 filed on 6 Sep. 2012.

SCOPE OF THE INVENTION

In one aspect, the present invention provides a transgenic non-humananimal model which is selected to overexpress Spy1A under the control ofa MMTV promoter causing the animal model to develop cancer, andpreferably breast cancer. In another aspect, the present inventionprovides a transgenic non-human animal model selected to express Spy1Ain one or more tissues thereof when an antibiotic is fed to the animal,and leading to the development of cancer, most preferably breast cancer.In another aspect, the present invention provides a transgenic non-humananimal model, preferably a mammal and most preferably a mouse model,which includes a MMTV-SV40-Spy1A gene sequence (hereinafter referredinterchangeably as “MMTV-SV40-Spy 1A”, “MMTV-SV40-Spy1”, “MMTV-Spy1A”and “MMTV-Spy1”). The animal model of the invention may permit uses inthe identification of agents for inhibiting or treating cancer, ornamely breast cancer.

BACKGROUND OF THE INVENTION

Amid all cancers known to afflict the Canadian population, breast cancer(BC) is documented as the second leading cause of cancer deaths amongfemales. Current knowledge of the molecular signatures and biochemicalpathways that govern BC initiation and progression is far fromcomprehensive and requires further expansion in order to identifyputative biomarkers that undoubtedly predict the correct therapeuticcourse of action to take with each patient. Due to the heterogeneousnature of cell types which cooperate to form a functional post-natalmammary gland, the various clinical forms of BC that may arise arecurrently distinguished based on prognostic criteria such ashistological phenotype, steroid and growth factor receptor status, andtumor ability to metastasize to neighbouring lymph nodes. In order tofully understand the various molecular mechanisms underpinning theevolution of mammary tumorigenesis, post-pubertal mammary glanddevelopment is often looked upon to highlight critical signalingpathways that possess the inherent capacity to mutate and/or becomederegulated in BC. Once maturity is established, the adult virginmammary organ retains the ability to cycle through four developmentstages: virgin, pregnancy, lactation, followed by involution andreversion to a virgin-like state. During early pregnancy-inducedlobuloalveolar development, elevated expression of prolactin, placentallactogens, and progesterone results in escalated rates of luminalepithelial proliferation, and promotes functional differentiation ofalveolar precursor cells into specialized structures proficient in milkrelease. Parturition-induced lactogenesis functions to nourish neonatesthrough alveolar milk production and secretion of colostrums intoenlarged luminal ducts. Neonate weaning initiates extensive luminalalveolar cell death (apoptosis) and epithelial remodelling duringinvolution, a process lasting for several days to allow forreinstatement of the mammary gland to a virgin-like appearance.

SUMMARY OF THE INVENTION

It has been appreciated that at a cellular level Speedy (Spy1A) plays arole in the DNA damage response, functioning to enhance cell survivaland promote cell proliferation in lieu of apoptosis. Spy1A is capable ofpromoting precocious development and tumorigenesis. Hence, determininghow Spy1 A protein levels are regulated may reveal novel informationregarding the dynamics of cell cycle control during normal and abnormalgrowth conditions. Furthermore, non-degradable forms of Spy 1 A do nottrigger intrinsic cell cycle checkpoints but, rather, promote cellproliferation and oncogenic cell transformation demonstration that thismechanism may contribute to tumorigenesis.

Further, it has been appreciated that Spy1A is a novel cell cycle genewhose product binds to cyclin-dependent kinase-2 (CDK2) and activatesits kinase activity to promote cell cycle progression through a cyclinindependent mechanism and to promote cell movement into DNA synthesis.Spy1A is expressed naturally at high levels in the proliferating mammarygland, and aberrant overexpression of Spy1A results in precociousmammary development and eventually tumorigenesis in vivo.

Spy1A elevation in c-Myc overexpressing tumors can be maintained duringprimary tumor culture, the MMTV-Myc mouse model, well documented for itsability to form aggressive mammary adenocarcinomas, may be utilized toderive a previously uncharacterized tumor cell line engineered tooverexpress c-Myc (henceforth referred to as F5A1-2).

Induction of the mammary oncogene c-Myc upregulates Spy1A and it isfurther demonstrated that Spy1A protein levels are elevated in mammarytissue and breast tumors derived from MMTV-Myc transgenic mice. Spy1Aknockdown in F5A1-2 cell lines led to downregulation of cyclin-dependentkinas inhibitors (CKI) p21 and p27, a 23% reduction in proliferationrate, and a shift in cellular phenotype to a spindle-like/fibroblasticmorphology. Together, findings support that Spy1A plays a functionalrole in mammary-related c-Myc signal transduction, and acts downstreamof ERα, c-Myc, and the MAPK cascade to regulate proliferation, mammarydevelopment, and carcinogenesis.

One possible non-limiting object of the present invention is to providea powerful tool for the study of cancer, namely breast cancer.Specifically, in one aspect the invention provides a transgenicnon-human animal model whose somatic cells contain at least one copy ofa MMTV-Spy1 transgene causing Spy1A overexpression under the control ofthe MMTV promoter, and causing this animal model to develop cancer, ormore particularly breast cancer. The animal is preferably hemizygous forthe transgene.

In another aspect, the present invention provides a transgenic non-humananimal model whose somatic cells contain a MMTV-Spy1A transgene whichcauses the animal model to develop cancer, or preferably breast or livercancer.

In yet another aspect, the present invention provides a transgenicnon-human animal model comprising germ cells and somatic cellscontaining an exogenous MMTV-SV40-Spy1A gene sequence introduced intosaid animal model or an ancestor of said animal model at an embryonicstage, wherein said gene sequence comprises a mouse mammary tumor virusgene (MMTV), a functionally disrupted SV40 gene (SV40) and a human Spy1Agene. It has been appreciated that a portion of an SV40 gene whenincorporated into a transgenic construct, or preferably theMMTV-SV40-Spy1A gene sequence increases expression or induceoverexpression of a gene under the control of a MMTV promoter.

In a preferred embodiment, the transgenic non-human animal comprisesgerm cells and somatic cells containing an exogenous MMTV-SV40-Spy1Agene sequence introduced into said animal or an ancestor of said animal,at an embryonic stage wherein said gene sequences comprises a mousemammary tumor virus promoter, a functionally disrupted SV40 gene and ahuman Spy1A gene.

Preferably, the animal model is hemizygous of the MMTV-SV40-Spy1A genesequence. The human Spy1A gene preferably includes a modified Spy1A geneof SEQ ID NO: 1 or a conservatively modified variant thereof. Mostpreferably, the MMTV-SV40-Spy1A gene sequence is introduced to theanimal model or the ancestor by microinjecting a fragment sequenceobtained from restriction enzyme digestion of SEQ ID NO: 5 or aconservatively modified variant thereof with XhoI and SpeI.

In yet another aspect, the present invention provides a transgenicnon-human animal model which is selected to express Spy1A in one or moretissues thereof when an antibiotic is fed to the animal model, saidexpression of Spy1A preferably leading to the development of cancerwithin said animal model, preferably breast or liver cancer.

In yet another aspect, the present invention provides a transgenicnon-human animal model whose germ cells and somatic cells contain anexogenous Spy1A-pTRE-Tight gene sequence (hereinafter interchangeablyreferred to as “Flag-Spy1A-pTRE”, “Flag-Spy1A-pTRE-Tight”,“Flag-Spy1-pTRE”, “Flag-Spy1-pTRE-Tight”, “Spy 1A-pTRE”, “Spy1A-pTRE-Tight”, “Spy1-pTRE” and “Spy1-pTRE-Tight”) introduced into theanimal model or an ancestor of the animal model at an embryonic stage,wherein said gene sequence comprises a human Spy1A gene.

Preferably, the animal model is hemizygous of the Spy1A-pTRE-Tight genesequence. The human Spy1A gene is preferably a modified Spy1A gene ofSEQ ID NO: 1 or a conservatively modified variant thereof. Mostpreferably, the Spy1A-pTRE-Tight gene sequence is introduced to theanimal model or the ancestor by microinjecting a fragment sequenceobtained from restriction enzyme digestion of SEQ ID NO: 18 or aconservatively modified variant thereof with XhoI and A1wNI.

In a preferred embodiment, the animal model is selected to express theSpy1A gene and develop cancer when administered with a tetracycline.Most preferably, the tetracycline is doxycycline. Preferably, the canceris breast or liver cancer.

The animal model of the present invention is not strictly limited tothose belonging to any specific genus or species, provided that theanimal model preferably permits introduction of exogenous geneticsequences to be incorporated into the genome. The animal model ispreferably a mouse, a rat, a monkey, a sheep, a dog, a rabbit, or ahorse. Most preferably, the animal model is a mouse or a rat.

In yet another aspect, the present invention provides a method ofproducing the transgenic non-human animal, the method comprisingmicroinjecting the fragment sequence into a fertilized embryo andtransplanting said fertilized embryo into a surrogate animal.

In yet another aspect, the present invention provides a tumor cell linecomprising a plurality of cells, wherein the cells are derived from theanimal model or comprise the fragment sequence.

In yet another aspect, the present invention provides a method forscreening an agent for treating or preventing cancer, the methodcomprising administering the agent into the animal model or the tumorcell line and detecting size reduction of a tumor caused by the cancer.Preferably, the cancer is breast or liver cancer.

In yet another aspect, the present invention provides a transgenicnon-human animal model comprising germ cells and somatic cells having anendogenous MTB-Spy1A gene sequence, wherein the animal model is aprogeny generated by crossing a MMTV-rtTA non-human animal model and theanimal model having the Spy1A-pTRE-Tight gene sequence derived from thefragment sequence obtained from restriction enzyme digestion of SEQ IDNO: 18 or a conservatively modified variant thereof with XhoI and A1wNI,the progeny being selected to express the Spy1A gene when administeredwith a tetracycline.

In yet another aspect, the present invention provides a transgenicnon-human animal model, the animal model being a progeny obtained frombreeding first and second ancestors, wherein the first ancestorcomprises respective germ cells and somatic cells having an ancestorgene sequence introduced into the genome of the first ancestor at anembryonic stage, the ancestor gene sequence comprising a promotersequence and a tetracycline transactivator (tTA) or reverse tetracyclinetransactivator (rtTA) gene sequence, and the second ancestor comprisesrespective germ cells and somatic cells having a Spy1A-pTRE-Tight genesequence introduced into the genome of the second ancestor at anembryonic stage, the Spy1A-pTRE-Tight gene sequence comprising a humanSpy1A gene.

In yet another aspect, the present invention provides a transgenicnon-human animal model comprising germ cells and somatic cells having aplurality of gene sequences introduced into the genome of said animalmodel or an ancestor of said animal model at an embryonic stage, whereina first one of said gene sequences comprises a promoter sequence and atetracycline transactivator (tTA) or reverse tetracycline transactivator(rtTA) gene sequence, and a second one of said gene sequences comprisesa Spy1A-pTRE-Tight gene sequence, the Spy1A-pTRE-Tight gene sequencecomprising a human Spy1A gene.

In a preferred embodiment, the first ancestor is female, and the secondancestor is male. In one embodiment, the animal model comprises germcells and somatic cells having the ancestor gene sequence and theSpy1A-pTRE-Tight gene sequence. In a further preferred embodiment, theanimal model is selected to express the Spy1A gene and develop cancer,or more preferably breast cancer, when administered with a tetracycline,or more preferably a doxycycline.

It is to be appreciated that the promoter sequence is not restricted toany particular promoter sequence. In one embodiment, the promotersequence is selected to induce transcription of the tTA or rtTA genesequence in a target organ. Non-limiting examples of the target organinclude the brain, heart, skin, liver, stomach, intestine, breast, andreproductive organs. In one embodiment, the promoter sequence comprisesa mouse mammary tumor virus (MMTV) gene, and the target organ is thebreast. In one embodiment, the ancestor gene sequence or the first genesequence comprises the rtTA gene sequence.

In one embodiment, the animal model or the second ancestor is hemizygousof the Spy1A-pTRE-Tight gene sequence, and the human Spy1A genecomprises a modified human Spy1A gene of SEQ ID NO: 1 or aconservatively modified variant thereof. In one embodiment, theSpy1A-pTRE-Tight gene sequence is introduced into the genome bymicroinjecting a fragment sequence obtained from restriction enzymedigestion of SEQ ID NO: 18 or a conservatively modified variant thereofwith XhoI and A1wNI.

Preferably, the conservatively modified variants have at least 70%, 75%,80%, 85%, 90%, 95% or 99% sequence identity to the specific referencednucleotide or amino acid sequence. The conservatively modified variantsmay include point mutations, as well as deletions, substitutions,insertions, transitions, amplifications, inversions, transversions orothers of one or more nucleotide bases or amino acid residues.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference may now be had to the following detailed description, takentogether with the accompanying drawings in which:

FIG. 1 shows a fusion gene fragment construct for producing a transgenicmouse according to an embodiment of the present invention.

FIG. 2 shows identification of positive founders confirmed through PCRanalysis. Positive founders are indicated by the presence of an 825 bpfragment.

FIG. 3 shows GAPDH (100 bp) control for the identification of positivefounders as shown in FIG. 2.

FIG. 4 shows identification of SDM-derived mutant Flag-Spy1A-pLXSNconstructs upon detecting of a 977 bp fragment following EcoRI digestionof isolated plasmid DNA from each colony: colony 1.1 (lane 1); colony1.2 (lane 2); colony 1.3 (lane 3); colony 2.1 (lane 4); colony 2.2 (lane5); and colony 2.3 (lane 6). The pLXSN vector backbone was estimated at7.0 kb, and the Spy1A insert was estimated at 1.0 kb (997 bp).

FIG. 5 shows EcoRI digestion of the MMTV-SV40-Spy1A transgenic vectorreleasing the flag-Spy1A coding sequence from the remaining vectorbackbone. EcoRI digestion of the resultant transgene DNC produced a 977bp fragment as expected and confirmed successful cloning. The pMMTV-SV40backbone was estimated at 6.0 kb and the flag-tagged Spy1A insert wasestimated at 1.0 kb (997 bp).

FIG. 6 shows digestion of MMTV-SV40-Spy1A prior to microinjection.

FIG. 7 shows detection of a single copy of MMTV-SV40-Spy1A DNA utilizingPCR genotyping methods. Transgene DNA was successfully detected using 8%PAGE in order to verify the success of using the MO23/MO23 primer setfor detection of the Spy1A transgene in tail clip samples. PCRamplification of MMTV-SV40-Spy1A vector DNA (lane 2) using M022/M023primers produced an 825 bp amplicon, identical to the positive MMTVvector control (+MMTV, lane 1) as expected.

FIG. 8 shows successful transmission of transgene from founder tooffspring using primer pair M022 (SEQ ID NO: 2)/M023 (SEQ ID NO: 4)resulting in a 825 bp fragment.

FIG. 9 shows confirmation of germline transmission of transgene usingprimer pair A933 (SEQ ID NO: 3)/M023 (SEQ ID NO: 4) resulting in a 197bp fragment.

FIG. 10 shows a Spy1-pTRE vector map according to an embodiment of thepresent invention.

FIG. 11 shows a restriction digest of Xhol and A1wNI for isolating aportion of the vector illustrated FIG. 10 for a subsequentmicroinjection step according to an embodiment of the present invention.

FIG. 12 shows identification of Spy1-pTRE founder mice via PCR analysisin the presence of a 536 bp band. The number labels correspond to mousetag numbers belonging to each tail sample screened, and the label“vector” corresponds to the Spy1-pTRE transgenic vector used as apositive control.

FIG. 13 shows confirmation of successful germline transmission ofSpy1-pTRE transgene according to an embodiment of the present invention.The number labels correspond to mouse tag numbers belonging to each tailsample screened, and the label “vector” corresponds to the Spy1-pTREtransgenic vector used as a positive control.

FIG. 14 shows a Flag-Spy1A-pTRE Tight vector map according to anembodiment of the present invention. Primers, Spy1 and pTRE promoter areoutlined.

FIG. 15 shows a linearized map of a Flag-Spy1A-pTRE Tight vectorindicating the locations of promoter, Spy1 and primers.

FIG. 16 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 0.5 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to pTRE vector control (447 bp); and the lanes “G”correspond to GAPDH (about 100 bp).

FIG. 17 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 1 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to pTRE vector control (447 bp); and the lanes “G”correspond to GAPDH (about 100 bp). The band of correct size is outlinedunder the lane “3P”.

FIG. 18 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 2 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to pTRE vector control (447 bp); and the lanes “G”correspond to GAPDH (about 100 bp). The band of correct size is outlinedunder the lane “3P”.

FIG. 19 shows a 4.25% polyacrylamide gel image of Spy1 -pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 3 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to pTRE vector control (447 bp); and the lanes “G”correspond to GAPDH (about 100 bp). The band of correct size is outlinedunder the lane “3P”.

FIG. 20 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 0.5 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to a maxi-prepped pTRE vector control with ahigher concentration; and the lanes “G” correspond to GAPDH. The band ofcorrect size is outlined under the lane “3P”.

FIG. 21 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 1 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to a maxi-prepped pTRE vector control with ahigher concentration; and the lanes “G” correspond to GAPDH. The band ofcorrect size is outlined under the lanes “1P” and “3P”.

FIG. 22 shows a 4.25% polyacrylamide gel image of Spy1-pTRE DNA samples1, 2 and 3 amplified with PCR using the primer combination A548/A549with 2 second exposure time. The lane “L” corresponds to a ladder; thelanes 1P, 2P and 3P correspond to samples 1, 2 and 3, respectively; thelane “CP” corresponds to a maxi-prepped pTRE vector control with ahigher concentration; and the lanes “G” correspond to GAPDH. The band ofcorrect size is outlined under the lanes “1P” and “3P”.

FIG. 23 shows a bar graph illustrating the results from a qRT PCRanalysis test for Spy1 overexpression in the mammary glands of aMMTV-Spy1 mouse in accordance with a preferred embodiment of theinvention, and which shows log 10 expression of Spy1 as the Y axiscompared to GAPDH.

FIG. 24 shows a DMBA treatment plan for a MMTV-Spy1 mouse and its pairmatched littermates, and which indicates age at beginning and endtreatment.

FIG. 25 shows a bar graph depicting the percentage of MMTV-Spy1 andcontrol mice (Y axis) that developed all tumour types, mammary tumours,and ovarian tumours.

FIG. 26 shows a line graph depicting the percentage of tumour free mice(Y axis) at the indicated ages in weeks (X axis).

FIG. 27 shows a bar graph depicting the percentage of MMTV-Spy1 and pairmatched littermates (F1 cntl) developing hepatocellular carcinoma 1 yearof age and older.

FIG. 28 shows a bar graph illustrating the results from a qRT PCRconducted on liver tissue collected from MMTV-Spy1 mice and their pairmatched littermates, and which illustrates Spy1 expression on a log10scale as compared to GAPDH.

FIG. 29 shows a bar graph illustrating the results from a qRT PCRconfirming Spy1 overexpression upon delivery of doxycycline to aMTB-Spy1 mouse generated by crossing a Spy1 -pTRE mouse with a MMTV-rtTAmouse.

FIG. 30 shows a breeding scheme for a male Spy1-pTRE mouse in accordancewith a preferred embodiment of the present invention and a femaleMMTV-rtTA mouse, and which illustrates possibly genotypes of theresulting progenies.

FIG. 31 shows a bar graph illustrating the results from a qRT PCRconfirming elevated Spy1 expression upon delivery of doxycycline to aMTB-Spy1 progeny mouse generated by crossing a male Spy1 -pTRE parentmouse with a female MMTV-rtTA parent mouse.

FIG. 32 shows a bar graph illustrating the results from a qRT PCRconfirming elevated Spy1 expression upon delivery of doxycycline to aMTB-Spy1 progeny mouse generated by crossing a male Spy1-pTRE parentmouse with a female MMTV-rtTA parent mouse, when compared to the Spy1expression of the parent mice.

FIG. 33 shows a bar graph illustrating the results from a qRT PCRconfirming elevated Spy1 expression in the mammary glands of a femaleMTB-Spy1 progeny mouse upon delivery of doxycycline thereto, and whichis generated by crossing a male Spy1-pTRE parent mouse with a femaleMMTV-rtTA parent mouse.

DETAILED DESCRIPTION OF THE INVENTION

The gene fragment construct MMTV-SV40-Spy1A (SEQ ID NO: 5) for thedevelopment of a transgenic mouse according to a preferred embodiment ofthe present invention is shown in FIG. 1. The construct was microinjected at roughly 4.7 kb into 357 fertilized embryos fromsuperovulated female mice and transplanted into pseudo pregnant CD-1female mice. This resulted in 43 pups being born of which 13 testedpositively for the MMTV-SV40-Spy1A as confirmed in the PCR analysisshown in FIGS. 2 and 3.

To prepare the MMTV-SV40-Spy1A construct, Flag-Spy1A-pLXSN containingthe complete coding sequence of the human Spy1A gene conjugated to aflag tag was provided. Site-directed mutagenesis (SDM) was utilized tocreate a second EcoRI site positioned near the terminal region of thehuman Spy1A coding sequence (SEQ ID NO: 1) in Flag-Spy1A-pLXSN forefficient removal of the intrinsic poly-A tail.

SDM primers A424 and A425 (SEQ ID NOs: 6 and 7) were designed to flankthe vector region targeted for mutation. SDM reactions were performedwith the following components: Flag-Spy1A-pLXSN vector DNA (10-100 ng);0.3 mM dNTP mix (Cat. No. DD0057, Biobasic Inc., Ontario, Canada); 1×pfx buffer and 1 μl pfx polymerase (Cat. No. 11708-013, Invitrogen,Canada); 1mM MgSO4; 1 μM each of A424 forward and A425 reverse primers(SEQ ID NOs: 6 and 7); filter-sterilize nuclease free water up to 50.0μl. Cycling conditions for SDM include (1) 2 minutes at 94° C., (2) 25cycles of 94° C. for 15 seconds, 55° C. for 30 seconds and 68° C. for 5seconds, and (3) 68° C. for 10 minutes. SDM reaction products were DpnIdigested for 2 hours at 37° C. (Cat. No. ER1701, Fermentas, Burlington,Ontario, Canada), and subsequently transformed utilizing TOP10 E.coliand plated onto 100 mg/ml Ampicillin plates. Select colonies werescreened for EcoRI insertion and were identified upon detection of a 977bp fragment following EcoRI digestion (for 20 minutes at 37° C. (Cat.No. FD0274, Fermentas)) of isolated plasmid DNA from each colony (usingQIAprep Spin Miniprep Kit (Cat. No. 27104, Qiagen, Mississauga, Ontario,Canada) as shown in FIG. 4. Successful EcoRI insertion was confirmedthrough sequencing for two colonies in particular, Is.1 and IIs.3,utilizing A210 and A211 sequencing primers (SEQ ID NOs: 8 and 9).Purified vector DNA from Colony IIs.3 was subjected to EcoRI digestion(Cat. No. ER0271, Fermentas), and produced two fragments at 7.0 kb(vector backbone) and 1.0 kb (Spy1A gene insert). Digestion productswere separated, and the appropriate 1.0 kb fragment was gel extractedusing the EZ-10 Spin Column DNA Gel Extraction Kit (Cat. No. BS354,Biobasic Inc.) and purified.

EcoRI digestion of 2 mg of MMTV-SV40-TRPS-1 vector DNA ensued for 1 hourat 37°, followed by the immediate removal of terminal phosphate groupsfrom digested ends utilizing incubation with calf intestinal alkalinephosphatase (Cat. No. EF0341, Fermentas) for 30 minutes at 37°.Phosphatase treatment was necessary in order to prevent the re-ligationof linearized vector DNA termini. Consequently, reaction products wereseparated, followed by gel purification of the resultant 6.0 kb fragment(MMTV-SV40 backbone) using the EZ-10 Spin Column DNA Gel Extraction Kit.Ligation of the Spy1A gene insert into the MMTV-SV40 backbone wasconducted using T4 DNA ligase (Cat. No. EL0017, Fermentas), and ligationreactions were subsequently transformed utilizing TOP 10 E. coli andplated onto 100 mg/ml Ampicillin plates. Select colonies were screenedfor EcoRI insertion and were identified upon detection of a 977 bpfragment following EcoRI digestion of isolated plasmid DNA from eachcolony as shown in FIG. 5. Successful cloning of the Spy1A codingsequence into the MMTV-SV40 vector backbone was confirmed throughsequencing. Sequencing primers A252, A253, A254, A255, A256, A257, A258and A259 (SEQ ID NOs: 10 to 16) were utilized in order to verify theintactness of all transgenic vector components.

The resultant transgenic vector was designated as MMTV-SV40-Spy1A andcontains an untranslated portion of the Ha-ras gene, in addition to anSV40 polyadenylation site. Bacterial sequences such as those found invector backbones have been noted to inhibit successful incorporation oftransgenic DNA into the mouse blastocyst genome. Thus, XhoI/SpeI doubledigestion (Cat. Nos. ER0691 and ER1251, Fermentas) of purified vectorDNA (30 mg per tube) ensued, and resulted in the production of twofragments: 4.7 kb (MMTV-SV40-Spy1A transgene) and 2.9 kb (remainingbackbone) as shown in FIG. 6. Two vials of XhoI/SpeI digested transgenicDNA were made available for microinjection into mouse blastocysts forsubsequent creation of the first MMTV-SV40-Spy1A transgenic mouse modelknown to date. Transgene detection of a single copy of MMTV-SV40-Spy1ADNA was tested utilizing the PCR conditions outlined for M022 and M023genotyping primers (SEQ ID NOs: 2 and 4) as shown in FIG. 7.

The resulting transgene fragment was sent to the University of WesternOntario Transgenic Facility to undergo pronuclear injections. Tailsamples from the resulting litters were received and DNA was extractedusing the Qiagen Puregene Core Kit A for mouse tails. Transgenedetection was accomplished using two sets of primers with two uniqueforward primers (M022 (SEQ ID NO: 2) and A933 (SEQ ID NO: 3)) and onereverse primer (M023 (SEQ ID NO: 4)). PCR cycling conditions consistedof (1) denaturation at 94° C. for 3 min, (2) denaturation at 94° C. for1 min, annealing at 55° C. for 2 min, elongation at 72° C. for 1 min and(3) a final elongation step at 72° C. for 3 min. Each 25 uL PCR reactionwas made using UBI HP Taq DNA polymerase (HPTAQ-01) and contained afinal concentration of 2 ng/uL of pure genomic DNA, 1× buffer, 2 mMMgSO₄, 0.2 mM dNTP, 0.5 mM forward primer, 0.5 mM reverse primer and0.025 U/uL Taq polymerase. Additionally, a final volume of 1% and 4%DMSO was added for primer pairs M022 (SEQ ID NO: 2)/M023 (SEQ ID NO: 4)and A933 (SEQ ID NO: 3)/M023 (SEQ ID NO: 4) respectively. PCRamplification resulted in an 825 bp and 197 bp amplicon for primers M022(SEQ ID NO: 2)/M023 (SEQ ID NO: 4) and A933 (SEQ ID NO: 3)/M023 (SEQ IDNO: 4) respectively as shown in FIGS. 8 and 9, respectively.

Expression levels of Spy1A was tested in the inguinal mammary glands of6 week old MMTV-Spy1A mice and their negative littermates via qRT PCRanalysis to ensure Spy1A was being overexpressed in the mammary gland ofthis mouse model system. Spy1A was found to be significantlyoverexpressed in the mammary glands of MMTV-Spy1A mice as compared totheir control littermates (FIG. 23). To test for increasedsusceptibility to mammary tumourigenesis, MMTV-Spy 1 A mice and theirnegative littermates were treated with 1 mg of7,12-dimethylbenzanthracene (DMBA) once a week for 6 consecutive weeksbeginning at 8 weeks of age via oral gavage. Treatment plan indicatingage during treatment and at end of study is illustrated in FIG. 24. Micewere monitored on a weekly basis for the development of mammary tumoursvia palpitation. MMTV-Spy1 A mice were found to develop significantlymore mammary tumours than their control littermates (FIG. 25).Additionally, MMTV-Spy1A mice developed tumours earlier than theircontrol littermates (FIG. 26).

When collecting male MMTV-Spy1 mice over the age of 1 year, it was notedthere was an increased incidence of liver carcinogenesis in theMMTV-Spy1 mice as compared to their negative control littermates (FIG.27). Liver tissue was collected from MMTV-Spy1 male mice 1 year of ageand older along with pair matched littermate controls and the livertissue was subjected to qRT PCR analysis to determine Spy1 expression inthe liver. Spy1 was found to be significantly overexpressed in MMTV-Spy1male mice as compared to littermate controls (FIG. 28).

In accordance with another preferred embodiment of the presentinvention, the fusion gene fragment construct Flag-Spy1A-pTRE-Tight (SEQID NO: 18) as illustrated in FIGS. 10, 14 and 15 were prepared. Inparticular, a Caspase3-pTRE-Tight vector was digested with EcoRI andPvuII to remove Caspase3. A 20 bp linker was then added to close thevector. Site directed mutagenesis was performed on a Flag-Spy1A-pLXSNvector to create an EcoRI restriction enzyme site to enable extractionof Flag-Spy1A from the vector. EcoRI digestion was subsequentlyperformed to remove Flag-Spy1A from the Flag-Spy1A-pLXSN vector. TheFlag-Spy1A fragment was then ligated into the pTRE-Tight vector.

Successful preparation of DNA fusion gene fragment construct sampleswere confirmed by PCR amplification with the primer combinationA548/A549 (SEQ ID NOs: 19 and 20) and polyacrylamide gel (as shown inFIGS. 16 to 22) as well as DNA sequencing. The bands of correct size areoutlined under the lanes “1P” and/or “3P” in FIGS. 17 to 22. All testedPCR samples were confirmed by DNA sequencing.

The Spy1-pTRE plasmid were restriction digested using Xhol and A1wNI toisolate a portion for subsequent microinjection into a fertilized embryofrom a superovulated female mouse. The digested portion was confirmed bygel electrophoresis as shown in FIG. 11. The digested portion wasmicroinjected into fertilized embryos from superovulated female mice andtransplanted into pseudo pregnant CD-1 female mice. Some resulting pupstested positive as confirmed and shown in FIG. 12. Successful germlinetransmission of the Spy1-pTRE transgene was confirmed as shown in FIG.13.

The mice having the Spy1-pTRE gene sequence was fed doxycycline toactivate expression of Spy1A. Development of cancer including breastcancer was experimentally confirmed.

In a separate study, selected Spy1A-pTRE mice found to lack inducibleoverexpression of Spy1A were nevertheless found to be suitable forpreparing overexpressing progenies. In a controlled study, selectedSpy1-pTRE mice found to be without inducible overexpression of Spy1Awere preferably crossed with MMTV-rtTA mice to generated a MTB-Spy1mouse model. It has been appreciated that such animal model may permitinducible overexpression of Spy1 A preferably after administration ofdoxycycline to their diet in the form of food pellets. Indeed,expression of Spy1 was induced by administering 2 mg/mL of doxycyclineat 5 weeks of age. Mammary glands were collected at 6 weeks of age fromMTB-Spy1, Spy1-pTRE and MMTV-rtTA mice for qRT analysis to test forincreased expression of Spy1 in the MTB-Spy1 mouse as compared to theselected control Spy1-pTRE and MMTV-rtTA mice. Spy1 was found to beoverexpressed in the MTB-Spy1 mouse, indicating this model system isfunctioning correctly (FIG. 29).

In an additional study, a male Spy1-pTRE mouse in accordance with apreferred embodiment of the present invention was crossed with a femaleMMTV-rtTA mouse received from a collaborator, and which is described inEdward J. Funther et al. “A novel doxycycline-inducible system for thetransgenic analysis of mammary gland biology”. The FASEB Journal. 16.3(2002): 283-292, the entire contents of which are hereby incorporated byreference. The female MMTV-rtTA mouse included the mouse mammary tumorvirus gene (MMTV) and a reverse tetracycline transactivator (rtTA), suchthat the MMTV promotor portion drives the expression of rtTA (‘Tet-On’).As illustrated in FIG. 29, four different genotypes were expected fromthe crossing, or namely a wild type progeny mouse, a Spy1-pTRE progenymouse, an MMTV-rtTA progeny mouse and the intended MTB-Spy1 progenymouse, the latter of which includes the transgenic elements from bothparent mice. It has been appreciated that the intended MTB-Spy1 progenymouse may permit for an inducible Tet-On system for expression of theSpy1 gene in the presence of a tetracycline, or preferably doxycycline,and as activated by the rtTA protein. As seen in FIGS. 31 to 33, qRT-PCRanalysis confirmed that upon exposure to doxycycline, a MTB-Spy1 progenyfemale mouse showed elevated Spy1 expression in the mammary glands whencompared to a control mouse, and the parent MMTV-rtTA and Spy1A-pTREmice.

The applicant has appreciated that the present invention providesvarious advantages and applications, and which include withoutrestriction a transgenic non-human animal model whose somatic cellscontain at least one copy of a MMTV-Spy1A transgene causing the animalmodel to develop cancer.

In yet another aspect, the present invention provides a transgenicnon-human animal model all of whose germ cells and somatic cells containan exogenous MMTV-SV40-Spy1A gene sequence introduced into said mammal,or an ancestor of said mammal, at an embryonic stage wherein said genesequence comprises a mouse mammary tumor virus gene (MMTV), afunctionally disrupted SV40 gene (SV40) and a modified human Spy1A geneof SEQ ID NO: 1.

Other applications of the invention include without restriction:

-   -   Methods of screening drugs/vaccines/or other vehicles developed        for the prevention of the development of cancer;    -   The study environmental factors and their effects on the        development of cancer;    -   The study cancer initiated at various stages of the animals        development;    -   Methods of screening drugs candidates and their        anti-carcinogenic;    -   Methods of screening drugs/vaccines/or other vehicles developed        for the prevention of the development of cancer;    -   The study environmental factors and their effects on the        development of cancer; and    -   The study of cancer namely breast cancer based on a novel        expression of Spy1A initiated within a model animal by feeding        the animal doxycycline.

Additional applications of the invention include, without restriction:

-   1. Expression of Spy1 A within one or more tissues of the model    animal is activated by the animal model ingesting doxycycline (Dox).-   2. The expression of Spy1A results in the tissues of the animal    model results in the development of cancer namely breast cancer    within that model animal.-   3. A transgenic non-human animal model in this case being a mouse    incorporates the condition and promoter response of claims 1 and 2.-   4. The mouse animal model is able to pass this condition expressed    in claims 1 and 2 along to subsequent generations when cross with a    mouse not having this condition.-   5. The transgenic non-human animal of claim 1, can be said animal    selected from the group consisting of mice, rats, monkeys, sheep,    and rabbits.-   6. Analysis of animal model DNA is able to confirm that transgenic    condition exists in said animal model.-   7. Transgenic animal model may be used to:    -   a. Study cancer    -   b. Study cancer initiated at various stages of the animals        development    -   c. Method of screening drugs candidates and there        anti-carcinogenic    -   d. Method of screening drugs/vaccines/or other vehicles        developed for the prevention of the development of cancer.    -   e. Study environmental factors and their effects on the        development of cancer

We claim:
 1. A method of producing a transgenic non-human animal modelcomprising germ cells and somatic cells having a Spy1A-pTRE-Tight genesequence introduced into the genome of said animal model or an ancestorof said animal model at an embryonic stage, the gene sequence comprisinga human Spy1A gene, wherein the method comprises microinjecting afragment sequence obtained from restriction enzyme digestion of SEQ IDNO: 18 or a conservatively modified variant thereof with XhoI and A1wNIinto a fertilized embryo and transplanting said fertilized embryo into asurrogate animal.
 2. The method of claim 1, wherein said animal model ishemizygous of said Spy1A-pTRE-Tight gene sequence, and said human Spy1Agene comprises a modified human Spy1A gene of SEQ ID NO: 1 or aconservatively modified variant thereof.
 3. The transgenic non-humananimal model of claim 1, wherein said animal is selected to express theSpy1A gene and develop cancer when administered with a tetracycline. 4.The transgenic non-human animal model of claim 3, wherein saidtetracycline is doxycycline.
 5. The transgenic non-human animal model ofclaim 3, wherein said cancer is breast cancer.
 6. The transgenicnon-human animal model of claim 1, wherein said animal model is selectedfrom the group consisting of a mouse and a rat.